Calibration method

ABSTRACT

The invention relates to a method of calibrating an optical system, the system comprising a flash lamp as a light source ( 4 ), a lens system ( 6 ), a monochromator comprising a grating ( 10 ), a motor ( 20 ) for displacing the grating so as to enable scanning essentially monochromatic light over a detection system. The light source provides at least two high intensity peaks at distinct wavelengths. The method comprises scanning a first wavelength region comprising at least one of said at least two high intensity peaks, and measuring the intensities at a selected number of points during the scan. A first of said at least two peaks is coarsely located. A wavelength region around each of said at least two peaks is scanned, measuring the intensities at closer intervals than previously. At least two peaks are located by autocorrelation. The location of said peaks is determined in terms of a distance from a reference point, said distance corresponding to said displacement of the grating.

The present invention relates to a method of wavelength calibration ofan optical system, and to selfdiagnosis of such a system comprising suchcalibration.

BACKGROUND OF THE INVENTION

Prior art spectrophotometer systems frequently make use of a deuteriumlamp as a light source. The advantage of such a lamp is that it burnsnicely in a continuous manner, and it has a couple of well defined wavelength peaks. However, the intensity of a deuterium lamp is relativelylow. In a liquid chromatographic system the small flow cells requireshigh intensity light in order to make adequate detection possible. Alsothe optical system comprising e.g. fibre optics require much more lightthan is delivered by a deuterium lamp. Wavelength calibration in suchsystems is performed by locating a single peak. This inevitably yieldsproblems of accuracy at the short wave and long wave ends respectivelyof the wave length scale. It is also very important that a selected wavelength be reproducible. The reproducibility reachable in calibrationshould be at least ±2 nm.

U.S. Pat. No. 5,212,537 is directed to a method of calibration formonochromators and spectrophotometers, using a continuous light source,a tungsten filament mercury vapour lamp, having two sharp intensitypeaks. The location of the peaks is identified by measuring the width ofsaid peaks at half height, and the midpoint of this width is taken asthe location of the peak. This technique would not be possible to use inconnection with a flash lamp, e.g. a Xenon lamp, since the intensity ofthe peaks fluctuates.

U.S. Pat. No. 5,268,737 is directed to calibration of aspectrophotometer. As the source of continuous light, a deuterium lampand a tungsten lamp is used. A point of reference is determined by usingzero order light. This would not be possible in connection with the useof optical fibres for transmission.

SUMMARY OF THE INVENTION

The object of the invention is to provide an improved calibration methodfor a spectrophotometer system, wherein the drawbacks of the prior artsystems are remedied.

This object is achieved with the method of the present invention.

The advantage of the method as claimed is that by virtue of the highbrightness of the flash lamp used in the method, the amount of lightintroduced in the system is increased, and that the accuracy in thecalibration is ascertained over the entire wavelength region ofinterest.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures

FIG. 1 is an overview of an optical system in which the invention may beimplemented;

FIG. 2 is a spectrum for a Xenon flash lamp; and

FIG. 3 schematically shows the mechanism for rotating the grating.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

In FIG. 1 there is shown an overview of a UV-visible detection system,generally designated 2, wherein the calibration method of the inventionis implemented.

The system comprises a light source, e.g. a xenon flash lamp 4, having atypical emission spectrum as shown in FIG. 2. In a preferred embodimentthe distance between electrodes in the lamp is approximately 1.5 mm.This yields a flash with very high brightness. The system furthercomprises lenses 6 that focus the light onto an entrance slit 8, apivotal, concave aberration corrected holographic grating 10 that causesa diffraction of the light. The grating has preferably 1200 lines/mm forthe applications in question, but may be selected by the skilled man tosuit a particular application. An optical fibre 12 is arranged in thearea where the light diffracted from the grating impinges, such that itis possible to select a wavelength of a certain narrow band width, forthe purposes of the invention in principle monochromatic light, and totransfer light of said wavelength to the detection system. By rotatingthe grating 10 around a pivot point P it is possible to select anywavelength from about 190 nm up to 700 nm. Rotation of the grating 10 isachieved with a stepper motor 20 (see FIG. 3) operating a micrometerscrew, schematically shown at 22. A nut means 23 running longitudinallyon the screw 22 is provided with a protruding element 25 that displacesa rod or arm 24 provided with a ball 27, and secured to the grating 10.The mechanics is designed such that a linear displacement of themicrometer screw 22 yields a linear displacement of the wavelength ofthe light beam impinging on the optical fibre 12. It is important thatthe longitudinal axis of the screw 22 is perpendicular to the bisector Bof the angle formed between the point of location of the entrance slit8, the pivot point P and the point of location of the optical fibre end13. This is a standard technical solution well known to the skilled man,and does not form part of the invention, and will thus not be discussedfurther herein.

Because the light source is a flash lamp, the light that is transferredinto the fibre needs to be split in a reference beam R impinging on areference detector 14, and a detection or sample beam S impinging on asample detector 16. The intensity of light from the reference beam Rimpinging on the reference detector is designated I^(o). The intensityof light impinging on the sample detector is designated I. The samplebeam enters a cell, e.g. a flow cell 18 through which samples are passedfrom a chromatographic column (not shown), ending up in a collectionsystem (not shown) for collecting fractions of eluted material, andimpinges on said sample detector 16 which detects the presence of samplein the flow cell 18 as a change in transmitted intensity I, and hence achange in absorbance A. Absorbance is defined in accordance with thewell known equation $A = {{- \lg}\frac{I}{I_{o}}}$

Absorbance A is proportional to concentration C, and path length 1 inthe cell according to following equation

 A=ε1C

wherein ε is the molar absorption coefficient.

Turning now to FIG. 2, the spectrum of the xenon lamp is shown. It is acontinuous spectrum at the high currents employed during discharge, andapart from a very broad peak at approximately 200 nm, there are a coupleof characteristic peaks, which are employed for calibration purposes.Thus the peak at 229.6 nm is used as one calibration wave length, andthe peak at 541.9 nm as the other. These peaks are in the first orderspectrum of the grating. For control purposes a third peak, namely thesecond order spectrum peak corresponding to 229.6 nm, i.e. 459.2 nm inthe first order, is used.

Now the calibration procedure will be described.

The first step is to run through the span of the micrometer screw 22between its end positions. The end positions are sensed by an opticalsensing device detecting the presence or absence of mechanical “flags”26 provided on the micrometer screw. This initial run is performed tocondition the micrometer and lubricate it by distributing the oil. Also,jamming of any kind will be detected thereby, and if occurring, amessage to that effect will be displayed to the operator, i.e. acalibration is not possible.

Secondly the wavelength field between approximately 100 and 352 nm willbe scanned, and one value for each nm, corresponding to three steps ofthe stepper motor, is detected and stored in a data array. The highestvalue of these is identified, and is designated max. peak. Themeasurement is performed as follows: After having stepped to a positioncorresponding to 100 nm (i.e. a number of steps from the end positiondetermined by the previously mentioned flag. This number of steps isapproximately known from experience and is preset), the stepper motorrests for a period long enough for the detection system to detect 5flashes from the xenon lamp. The lamp flashes continuously at a rate of100 Hz, thus 5 flashes would require the stepper motor to rest 50 msbefore it goes on. The intensities of said five flashes are added andstored as one entry in the data array. Of course the number of flashesdetected need not be five, but in order to obtain a reasonable averagingof variations, five has been selected as a good compromise.

After detection of said five intensity values, the stepper motor stepsthree steps, corresponding to 1 nm in a few microseconds and then restsagain for the system to detect five new intensity values, adding thesame and storing them in the data array. This is repeated over the range100 to 352 nm. Because of the system optics absorbs light in the UVregion, the system will not “open” until around 190 nm, and thus thedetectors will see no light until then.

After the scan is completed, the first detected intensity value abovezero stored in the data array (i.e. a value at approximately 190 nm orso) is compared to the highest detected value. If said first value isless than 2% of highest value (max. peak) the system proceeds. If it isnot, an indication to that effect will be displayed, signalling somekind of malfunction, normally defective optics. For example stray lightcould cause a background level above zero. The value 2% is arbitrarilyselected as a reasonable measure that the optics is functioningproperly. If the value is higher, there may be artefacts such asreflections from foreign particles etc. The 2% value is not critical andcould in principle be as high as 20%. The value at the “2% point” istaken as the starting point for the next step of the method.

In the next step the system checks the intensity values in the dataarray in order to find the peak at 229.6 nm. However, the broad peek atapprox. 200 nm mentioned above may in some instances, e.g. extremelyhigh discharge currents, have a higher peak intensity than the peak at229.6 nm, and therefore the system is programmed to step in the dataarray to values corresponding to a value located at 36 nm (108 steps)from the “2% point” in order to pass said broad peak.

By comparing the data set by autocorrelation against an ideal peakcorresponding to a y=x² curve, the peak at 229.6 nm is located.Autocorrelation is a standard technique comprehensively described in theliterature, and reference is e.g. made to a compendium “Tidsserieanalys”by Lennart Olbjer, University of Lund, for more details and alternativemethods.

Thereafter the region between 225-235 nm is scanned again, now recordingone set of measurements for each step of the stepper motor, each stepcorresponding to approximately 0.33 nm, in order to find the peak at229.6 nm. Again autocorrelation, using the data in the data arraystarting at 225 nm, is used to find the exact location of the peak at229.6 nm. The region 535-565 nm is scanned in the same way, in order tofind the peak at 541.9 nm using the same autocorrelation method. Ofcourse it is conceivable to scan the entire spectrum, but in order tosave time scanning is restricted to two regions of the spectrum.

When the peaks have been established the intensity at 229.6 nm ischecked to see whether it exceeds a selected minimum value or not.

If not, an error message will indicate “Low light intensity”. This is anindication that the lamp should be replaced. The system also logs thetime the lamp has been in use, and after a certain number of hours amessage will be displayed saying that the lamp should be replaced.

The calibration constant is calculated. It is defined as the distance(counted in steps of the stepper motor) from the “flag” on themicrometer screw to the “knee” at the “2% point” on the intensity curve.The calibration constant is taken as an indication that the system isfunctioning properly if it is within certain predetermined limits. Thedispersion is also calculated. Dispersion is defined as number ofsteps/nm, and is calculated as${dispersion} = \frac{{number}\quad {of}\quad {steps}\quad {between}\quad {calibration}{\quad \quad}{peaks}}{{541.9\quad {nm}} - {229.6\quad {nm}}}$

The dispersion varies from batch to batch of the gratings and depends onthe tolerances of the mechanical system. Usable dispersions are 2,9-3,1steps/nm.

In another aspect of the invention, there is provided a check of thecalibration, i.e. a validation check to see if the calibration is stillvalid. Such a check may be necessary if the results of a chromatographicrun for some reason seem to exhibit artefacts. Normally the system“flips” between three wavelengths during a run, and it may occasionallyhappen that the stepper motor jumps past a step, because of jamming onthe micrometer screw etc. This will lead to a displacement of thecalibration setting.

The calibration check is performed by running the calibration procedureagain, but this time only calculating the deviation in the calibrationconstant and the dispersion established at the calibration. If there isa deviation the system will display a message to the operator, who thencan decide to recalibrate.

In this process there is an extra check against the second orderspectrum peak of the peak at 229.6 nm, which is found at 459.2 nm. Thispeak is blocked during a chromatographic run by inserting a UV blockfilter at 360 nm and higher.

During start-up other checks are performed, such as a lamp intensitycheck. If the check indicates low intensity, a warning message will bedisplayed indicating a need to change the lamp. The operation time forthe mechanics is also logged by the system and thus it willautomatically be displayed when it is probable that the mechanicalsystem needs service.

What is claimed is:
 1. A method of calibrating an optical system, thesystem comprising a flash lamp as light source (4), a lens system (6), amonochromator so as to enable scanning essentially monochromatic lightover a detection system (14, 16), the light source providing at leasttwo high intensity peaks at distinct wavelengths, the method comprisingthe following steps: i) scanning a first wavelength region comprising atleast one of said at least two high intensity peaks, and measuring theintensities at a selected number of points during the scan; ii) coarselylocating said at least two peaks; iii) scanning a wavelength regionaround each of said at least two peaks, measuring the intensities atcloser intervals than in step i); iv) locating said at least two peaksby auto correlation; and v) determining the point of location of saidpeaks as a distance from reference point.
 2. The method as claimed inclaim 1, wherein said monochromator comprises a grating (10) and a motor(20) causing a displacement of the grating, and wherein said distancecorresponds to the displacement of the grating.
 3. The method as claimedin claim 1, wherein the measured intensity values are stored in a dataarray.
 4. The method as claimed in claim 3, wherein a plurality ofmeasurements is made for each of said points, the sum of said pluralityof values being stored in said data array.
 5. The method as claimed inclaim 3 or 4, wherein said autocorrelation for locating the respectivepeaks is performed on the data in said data array.
 6. The method asclaimed in claim 1, wherein an array point corresponding to a firstdetected intensity value, lower by a preselected percentage of a highestdetected intensity value is taken as a starting point, and wherein saidfirst peak is located by stepping to a data point in said arraycorresponding to a wave length value 36 nm higher than said pointcorresponding to said first intensity value above zero.
 7. A method ofcalibrating an optical system, the system comprising a flash lightsource, a lens system, an entrance slit, a monochromator in the form ofa concave reflection grating, a motor for displacing the grating in apivoting fashion so as to enable scanning essentially monochromaticlight over an optical fibre end, the optical fibre being connected to adetection system, the light source providing at least two high intensitypeaks at distinct wavelengths, the method comprising the followingsteps: scanning a first wavelength region comprising at least one ofsaid at least two high intensity peaks, and measuring the intensities ata selected number of points during the scan; registering the highestintensity value; comparing the first detected value above zero with saidhighest intensity value in order to establish that it does not exceed apredetermined fraction of said highest value; finding the first valueexceeding the value corresponding to said predetermined fraction of saidhighest value; locating a first of said at least two peaks, byautocorrelation; scanning a wavelength region around each of said atleast two peaks, measuring the intensities at closer intervals than inthe previous scan; locating said at least two peaks by autocorrelation.